The present invention concerns the use of angiostatin and its active fragments to treat all the pathologic conditions involving an excessive or deregulated activation of leucocytes of myeloid origins, or where it is desirable to inhibit the chemotactic migration or the release of active chemical species. These conditions are mainly reportable to inflammatory conditions of different origins.
Angiostatin is a circulating proteolytic cleavage product of plasminogen that can suppress the growth of a variety of tumors through an as yet not completely defined mechanism. Several studies have proposed that angiostatin induces selective inhibition of endothelial cell proliferation, migration, induction of endothelial cell cycle arrest and apoptosis [Cao, 1996 #2497; Lucas, 1998 #2499; Lu, 1999 #2498; Griscelli, 1998 #2238].
Even if the obvious target of anti-angiogenic compounds are endothelial cells, an increasing body of evidence indicates that cells from the natural immune system such as monocyte/macrophages are important regulators of angiogenesis [Sunderkotter, 1994 #1138; Polverini, 1997 #1963]. Granulocytes, whose role is frequently underestimated, also appear to play a primary role in the initial phases of the angiogenic process [Cassatella, 1999 #2671; Kasama, 2000 #2670]. Recent studies on the HIV-Tat protein have shown that the angiogenic potential of this molecule is linked not only to VEGF mimicry, but also to recruitment of granulocytes in vitro and in vivo via a chemokine-like domain of this molecule [Benelli, 2000 #2680].
Although the precise role of neutrophils in tumor development is still uncertain as their contribution may be time-restricted, they could induce tumor growth by direct promotion of vessel generation under both physiological (wound healing) and pathological (cancer, psoriasis) conditions. To date, studies on the effects of angiostatin have been limited to vascular cells (endothelial and smooth muscle cells); its role in phagocyte control has never been investigated.
The inventors have found that angiostatin is a strong inhibitor of neutrophil migration, activation and degranulation, as well as of neutrophil-mediated angiogenesis.
Angiostatin reduced by about 20-30% the migration of primary monocytes towards chemotactic stimuli as fMLP, MCP-1 and HIV-Tat, but was much more efficient as granulocyte inhibitor. The activity of angiostatin on PMN has been confirmed by dose-dependence studies using chemoattractants as IL-8 (50 ng/ml), MIP-2 (200 ng/ml), Tat (1 xcexcM) or fMLP (10xe2x88x927M) able to induce a high chemotactic response (FIG. 1). The addition of angiostatin caused a strong inhibition of migration peaking at the concentration of 1.25-2.5 xcexcg/ml (pxe2x89xa60.0053 Student T test as compared to controls). Higher doses of angiostatin (5-10 xcexcg/ml) were less effective. Also the chemotaxis induced by the phorbol ester TPA (200 ng/ml) was dose-dependently inhibited by angiostatin (FIG. 1). The inhibition of TPA induced migration suggests a possible modulation of the MAP-kinase pathway downstream PKC (28), a signal transduction pathway known to be involved in granulocyte migration. Several different angiostatin preparations have shown the same biological properties (FIG. 1). On the contrary, intact plasminogen has not shown any inhibitory potential.
Additional studies on PMN migration have been performed using GRO-alpha and MIP-2 as chemoattractants, as these chemokines selectively engage the CXCR2 (while IL-8 targets both CXCR1 and CXCR2). Even in these experimental settings angiostatin showed an inhibitory activity on granulocyte migration, according to the doses and responses observed with the other chemoattractants.
As a further proof to in vitro data, the inventors have tested angiostatin in the matrigel sponge model in vivo (29). Previous observations indicated that the majority of growth factors do not act only on endothelial cells, but in most cases (i.e. HIV-Tat, VEGF and Kaposi sarcoma cells supernatants) are able to recruit also mesenchymal cells and phagocytes in matrigel implants (23,30). The addition of IL-8 along with matrigel causes a strong angiogenic reaction, characterized by abundant dilated neovascular formations, and granulocyte infiltration. The addition of angiostatin to the sponges, in addition to a total, block of the angiogenesis induced by IL-8, MIP-2, GRO-alfa or LPS (FIG. 2b), drastically reduced the total number of cells inside the implant, where, instead of vessels, can be observed only small lacunae without endothelial lining (FIG. 3b,f,h). Using an anti-myeloperoxidase antibody the inventors have also shown that leucocytes infiltrating the IL-8 containing gels are neutrophil granulocytes and monocytes (FIG. 3c). The invasion of degranulating neutrophils, observed by electron microscopy analysis, often precedes and leads the monocyte and endothelial cell invasion of the sponge (23,30). The fundamental role of neutrophils in angiogenesis induction has been confirmed in neutrophil-depleted mice, where matrigel implants enriched with chemokines are not vascularized (FIG. 2a).
The neutrophil degranulation observed in matrigel sponges (23,30) suggests an acivated phenotype for these cells during the neoangiogenic process. Angiostatin (MTT assay) can inhibit by 80% the mitochondrial activity of IL-8 stimulated granulocytes and by 63% the fMLP induced activation (FIG. 4b). Membrane-associated ATP synthase and angiomotin have been respectively indicated as putative angiostatin receptors on endothelial cells, the RT-PCR of these receptors on PMN and on other cells of the myeloid lineage have shown the expression of both receptors. ATP-synthase has also been detected by FACS analysis of neutrophils by a specific antibody. Consequently these receptors can mediate the observed inhibitory effects (FIG. 4a).
Angiostatin has also been successfully used to block HIV-Tat induced PMN recruitment and angiogenesis. The full-length 1-101 amino-acid HIV-1 Tat protein induces a strong neutrophil chemotactic response in vitro. The addition of angiostatin to the neutrophils in the upper chamber of the microwells caused a significant decrease in cell migration to Tat (FIG. 5). This inhibition appeared again to be strictly dose dependent, with a maximal activity at 2.5 xcexcg/ml. HIV-Tat harbors several active domains interacting with cell surface receptors, among these the RGD sequence which binds avb3 and avb5 integrin (7,8), the basic domain which binds and activates flk-1/VEGFR2 (15) and the cysteine-core domain with chemokine-like activity. To examine the role of the chemokine-like properties of Tat in the inhibitory activity of angiostatin, a peptide containing only the chemokine-like domain of Tat was used (CysL24-51). Similar to what observed with whole Tat, the CysL24-51 peptide strongly induced neutrophil migration which was again inhibited by angiostatin (FIG. 5). A maximal level of activity at 2.5 xcexcg/ml was observed, and again higher or lower doses showed a reduced effect.
The inclusion of Tat in matrigel sponges injected in vivo results in a strong, rapid angiogenic response (FIG. 5 lower panel). The addition of angiostatin along with Tat completely abolished the angiogenic response to Tat, bringing the hemoglobin levels in the gels (a quantitative indicator of vascularization) to baseline levels. Angiostatin alone was not angiogenic. Histological analysis of the implants showed a strong, hemorrhagic angiogenesis with massive cell infiltration in the implants with Tat, while Tat implants also containing angiostatin had low cellularity and no vessels.
All these experimental evidences demonstrate that angiostatin, besides the well known antiangiogenic activity, can reduce granulocyte recruitment and activation, reducing the local production and release of reactive oxigen species.
Accordingly, object of this invention is a method for treating a pathologic condition in which the recruitment and/or the activation of myeloid leucocytes, especially polymorphonucleate granulocytes, are involved, which comprises administering to an animal, preferably a human subject in need of such a treatment, an effective amount of angiostatin or a biologically active fragment thereof.
The term xe2x80x9cbiologically active fragmentsxe2x80x9d indicates peptides derived from angiostatin, which could be modified in the sequence by substitution or addition of one or more amino acids with other natural or synthetic ones, maintaining or improving the biological and pharmacological properties of the full length protein. The active fragments will preferentially contain the first three xe2x80x9ckringlesxe2x80x9d of angiostatin.
According to a preferred embodiment of the invention, angiostatin or active fragments thereof are used in the treatment of inflammatory pathologies.
The dose schedule of angiostatin according to this invention will be adapted to the pathology, the clinical condition of the patient, and to the route of administeration. As a general indication, the compounds will be used in dosages ranging from 0.5 mg/Kg to 500 mg/Kg, preferably 1 to 100 mg/Kg, more preferably from 2 to 50 mg/Kg, once or twice a day. The route of administration for the present invention include parenteral (subcutaneous, intramuscular, intravenous, intradermic), oral, nasal, rectal, ophtalmic and topic applications. The drug will be administered as solution, suspension, infusion, capsule, tablet, injectable, spray, creme, emulsion, gel, drops, and can be prepared by conventional procedures, for example as described in Remington""s Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, XVII Ed.